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The gene for CDK2NA generates several transcripts/proteins which differ from each other in their first exons. Three of these transcripts are generated by alternative splicing (isoform 1 a.k.a p16INK4A, isoform 2 and isoform 3 a.k.a p12), two of which are known to function as inhibitors of CDK4 kinase. One other transcript that is generated from this gene contains an alternate reading frame (ARF), with the first exon located 20kb upstream of the remainder of the gene(isoform 4 a.k.a. p14ARF, p19ARF, ARF). In spite of the structural and some functional differences, all the proteins encoded by the CDKN2A gene are involved in cell cycle G1 control.

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ICC/IF image of ab11048 stained human MCF7 cells. The cells were 4% PFA fixed (10 min) and then incubated in 1% BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab11048, 1 µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue). This antibody also gave a positive IF result in HeLa, Hek293 and HepG2cells.

Flow Cytometry - Anti-CDKN2A/p14ARF antibody [ARF 4C6/4] (ab11048)

Overlay histogram showing HeLa cells stained with ab11048 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab11048, 0.5µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG2a [ICIGG2A] (ab91361, 1µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed.

Thank you for contacting Abcam Technical Team and for taking the time to provide some useful details of the experiments. I am very sorry to hear that your customer is having problems with this product.
According to Swiss-Prot, the localization of the staining (CDKN2A/p14ARF) could be both cytoplasmic and nuclear. You may wish to take a look at this site for further information:
http://www.uniprot.org/uniprot/P42771
U2OS is a human osteosarcoma cell line; it is known that CDKN2A/p14ARF is widely expressed but not detected in certain tissue such brain or skeletal muscle. There are at least 4 different splice variants and the expression pattern of these isoforms (P42771-1, P42771-2, P42771-3, Q8N726-1) may vary in different tissue/cell types.
After reading through the detailed protocol you kindly forwarded to Abcam, I would like to make the following comments/suggestions:
1) Try to use cells which are intensively dividing and the cell culture should in exponential (log) phase of growth.
2) It may be worth staining HeLa cells or sections of human liver or lymphomas as good positive control.
3) In order to increase the signal, it would be worth applying ABC signal enhancing system.
I hope this helps and if I can assist further, please do not hesitate to contact me.

Thank you for your enquiry. Our source for this antibody has told me that ab11048 does detect endogenous p14ARF; please see the following publication for additional information:
Llanos S et al. Stabilization of p53 by p14ARF without relocation of MDM2 to the nucleolus. Nat Cell Biol 3:445-52 (2001). PubMed: 11331871
If you have any additional questions, please contact us again.

Thank you for your enquiry. Although I was unable to find out exactly what the originator used as a positive control, they have said that Hela cells should be appropriate to use as a positive control for this antibody. If you have any more questions, please contact me again.